Obligatory myocardial ischemia/reperfusion (I/R) associated with cardiac surgery induces an inflammatory response, leading to over-expression of proinflammatory cytokines and chemokines. These factors exacerbate post-ischemic cardiac dysfunction. Thus, regulation of this inflammatory response is critical for myocardial protection in cardiac surgery. However, the signaling mechanisms involved in this response remain unclear. While Toll-like receptor 4 (TLR4) regulates cellular inflammatory response to endotoxin, it is presently unknown whether this innate immunoresponsive receptor plays a role in cardiac inflammatory response to I/R. Our preliminary studies found that the production of inflammatory mediators following I/R is abrogated in heart lacking functional TLR4 and that this reduction of inflammatory mediator production correlates to a marked improvement in post-ischemic cardiac functional recovery. It is likely that endogenous agents generated and/or released during I/R activate myocardial TLR4 signaling pathways. In our preliminary studies, we found that proteins of approximately 50 to 100 kD in coronary effluent from post-ischemic heart are proinflammatory. Among these proteins, we identified heat shock cognate protein 70 (HSC). Furthermore, we found that neutralization of extracellular HSC with a polyclonal antibody suppresses TLR4-dependent myocardial inflammatory response and that HSC, when applied extracellularly, interacts with TLR4 and induces cytokine production in isolated macrophages in a TLR4-dependent manner. We hypothesize that TLR4 signaling is involved in post-ischemic cardiac inflammatory response and that extracellular HSC functions as an endogenous ligand to activate TLR4 signaling cascade. Investigation of myocardial TLR4 signaling during I/R and determination of the molecular mechanisms through which I/R activates TLR4 signaling cascade are the major goals of the proposed studies. The Specific Aims of this project are: 1) to define the role of TLR4 signaling in post-ischemic myocardial inflammatory response and to determine the contribution of HSC to activation of myocardial TLR4, 2) to determine the mechanisms through which the myocardium releases HSC, and 3) to determine the effect of HSC on TLR4 signaling pathways. The proposed studies should provide novel insights into the signaling mechanisms involved in myocardial innate immunoresponse to I/R and could suggest new therapeutic targets and approaches for improving outcome following cardiac surgery with obligatory I/R.